Abstract Zika virus (ZIKV) represents a new threat to global health, with particular relevance to the developing brain and risk of congenital microcephaly. The basis by which ZIKV leads to neuronal death in the brain of the fetus is unknown, but presumably relates to effects of ZIKV proteins on cellular health. ZIKV is an RNA Flavivirus encoding 10 proteins including the NS2B/NS3heterodimer that formsanactive serineprotease involved in cleavage of the ZIKV polypeptide intoindividualproteins. Here we propose to study the molecular basis of ZIKV-induced microcephaly. We hypothesize that the ZIKV protease can function to cleave cytosolichost cellular proteins in neural precursor cells, leading to a range of cellular defects,contributingtotoxicity.Thisapplicationbringstogetherthreeexperiencedresearchgroupswithatrackrecordof collaborationonZIKV.OurpublishedworkhasfocusedontropismofZIKVforneuralprecursorcells,interactionofZIKV with the immune system, small molecule inhibitors of ZIKV, and effect of ZIKV on neuronal survival. We have also published on neural stem cell biology, recessive human genetic microcephaly mutations, and flavivirus molecular pathology. Ourpreliminarydata demonstrates thatZIKV NS2B/NS3misexpression can mediate celldeath, in amanner similar to ZIKV-induced cell death. Cell death can be abrogated with a protease-inactive mutation or by inhibition of protease activity through ZIKV protease inhibitors (ZPIs). Proteomic analysis identified a series of proteins bound to the ZIKV protease, which we term the ZIKV Protease-Ome (ZPO), at least oneof which is adirect cleavage target of ZIKV NS2B/NS3, and leads to blocked cytokinesis and cell death when inactivated, in a manner similar to ZIKV-induced cell death. The goal of this application is to discover the molecular, cellular and genetic basis by which ZIKV proteins mediate neuronal cell death, particularly in the context of mammalian cerebral cortical development. We combine mass spectrometry analysis, protein interaction networks, advanced bioinformatics, neuronal culture models, analysis of cell cycle dynamics, and in vivo modeling. Importantly, we will focus on differences between toxicity observed with ZIKV proteins and other sequence-similar flaviviruses that do not mediate neuronal death, and will study the mechanisms of theparticularneuronalvulnerabilitytocelldeathfollowingZIKVexposure.Wewill:1]TestZPOconstituentstodetermine which are direct protease targets of ZIKV NS2B/NS3. 2] Test ZPO to determine mechanisms by which ZIKV NS2B/NS3-mediated cleavage leads to neuronal death. 3] Test the short and long term effects of ZIKV protease expression on the developing brain, and the degree of rescue by inhibition of the ZIKV protease. The goal of these experiments is to determine how specific components of the ZIKV genome lead to disrupted cortical development, to determine mechanisms by which ZIKV impairs cell survival, and whether medications might benefit women exposed to ZIKVduringfetalbraindevelopment.